Hearing loss is primarily caused by damage to sensory hair cells (HC) in the cochlea of the inner ear. Humans and other mammals cannot replace damaged HCs;however, chicken, fish and amphibians can, by proliferation and transdifferentiation of neighboring supporting cells (SC) (Conwin and Oberholtzer, 1997). Interestingly, the tumor suppressor gene, p16INK4a, is absent in non-mammalian vertebrates that have the capacity to regenerate HCs (Kim et al., 2003;Gil and Peters, 2006). In mammals, p16INK4a acts as a cyclin-dependent kinase inhibitor that keeps cells in a quiescent state. Its expression is induced by age as well as mitogenic signaling (Zindy et al., 1997). p16INK4a is also known to play a critical role in the in vivo regenerative ability of adult cells, including neurons (Janzen et al., 2006;Molofsky et al., 2006). It is commonly thought that damaged HCs release signals that cause SCs to reenter the cell cycle. In non-mammalian vertebrates this results in HC regeneration;however, in mammals, we predict that this mitotic activity induces the expression of p16INK4a which keeps SCs in a quiescent state and thus prevents HC regeneration. Is it possible to give a mouse, the chicken's ability to regenerate HCs by inactivating p16INK4a? To achieve this goal, we propose the following central hypothesis: Inactivation of p16INK4a in mammals will allow SCs: (1) to respond to signals released from HCs damaged by antibiotics or genetic ablation, (2) to reenter the cell cycle and (3) to regenerate HCs. To test this hypothesis, we propose the following specific aims: Specific Aim 1: To determine the regenerative capacity of SCs in p161NK4a-null mice after HC damage caused by the aminoglycoside antibiotic, gentamicin. Specific Aim 2: To determine the regenerative capacity of SCs in p16INK4a-null mice after HC damage caused by acute inactivation of the retinoblastoma protein in postnatal HCs. Although mammals cannot spontaneously regenerate HCs, a process of HC regeneration similar to what occurs in non-mammalian vertebrates could be induced by genetic and/or therapeutic manipulations of cochlear SCs. This proposal is the first step toward the final goal of restoring hearing in those exposed to ototoxic drugs, loud noise or other environmental agents. In addition, HC damage induced by acute inactivation of the retinoblastoma protein in postnatal HCs is a technological breakthrough for the field.